Combined use of PRAME inhibitors and HDAC inhibitors

ABSTRACT

The invention relates to the cancer antigen PRAME (PReferentially expressed Antigen in MElanoma) and its use in a method of treatment of a tumour which comprises administering to a subject in need of treatment an effective amount of an inhibitor of PRAME, in combination with a second agent selected from the group of an inhibitor of HDAC (an HDACi) and a retinoid.

This application is the US national phase of international applicationPCT/EP2005/000937 filed 27 Jan. 2005, which designated the U.S. andclaims benefit of GB 0401876.8, filed 28 Jan. 2004, the entire contentsof each of which are hereby incorporated by reference.

The present invention defines a new use for the known cancer antigenPRAME (PReferentially expressed Antigen in MElanoma), and in particularprovides methods for the stratification of cancer patients based onPRAME expression and for the treatment of subjects with cancer by theinhibition of PRAME expression or activity.

Retinoic acid (RA) induces proliferation arrest, differentiation andapoptosis in a wide variety of cell types (Altucci, 2001; Freemantle,2003). Defects in retinoic acid receptor (RAR) signalling, such as thosecaused by the PML-RARα and PLZF-RARα translocations in acutepromyelocytic leukaemia, have been implicated in cancer (Altucci, 2001;Freemantle, 2003. Binding of RA to its receptor leads to release ofco-repressor molecules and recruitment of co-activators to RAR,resulting in activation of transcription (Xu, 1999).

Acetylation and deacetylation of histones is associated withtranscriptional events leading to cell proliferation and/ordifferentiation. Regulation of the function of transcription factors isalso mediated through acetylation. Recent reviews of histonedeacetylation include Kouzarides, 1999 and Pazin et al., 1997.

The correlation between the acetylation status of histones and thetranscription of genes has been known for over 30 years (see, forexample, Howe et al., 1999). Certain enzymes, specifically acetylases(e.g., histone acetyltransferase, HAT) and deacetylases (e.g., histonedeacetylase, HDAC), which regulate the acetylation state of histoneshave been identified in many organisms and have been implicated in theregulation of numerous genes, confirming the link between acetylationand transcription. See, for example, Davie, 1998. In general, histoneacetylation correlates with transcriptional activation, whereas histonedeacetylation is associated with gene repression.

A growing number of histone deacetylases (HDACs) have been identified(see, for example, Ng and Bird, 2000). The first deacetylase, HDAC1, wasidentified in 1996 (see, for example, Tauton et al., 1996).Subsequently, two other nuclear mammalian deacetylases were found, HDAC2and HDAC3 (see, for example, Yang et al., 1996, 1997, and Emiliani etal., 1998). See also, Grozinger et al., 1999; Kao et al., 2000; and Vanden Wyngaert et al., 2000.

HDACs function as part of large multiprotein complexes, which aretethered to the promoter and repress transcription. Well characterisedtranscriptional repressors such as Mad (Laherty et al., 1997), pRb(Brehm et al., 1998), nuclear receptors (Wong et al., 1998) and YY1(Yang et al., 1997) associate with HDAC complexes to exert theirrepressor function.

The study of inhibitors of histone deacetylases indicates that theseenzymes play an important role in cell proliferation anddifferentiation. The inhibitor Trichostatin A (TSA) (Yoshida et al.,1990a) causes cell cycle arrest at both G1 and G2 phases (Yoshida andBeppu, 1988), reverts the transformed phenotype of different cell lines,and induces differentiation of Friend leukaemia cells and others(Yoshida et al., 1990b). TSA (and SAHA) have been reported to inhibitcell growth, induce terminal differentiation, and prevent the formationof tumours in mice (Finnin et al., 1999).

The activity of HDACs and HATS (Histone Acetyl Transferases) isfrequently deregulated in cancer and one of the ways in which theseenzymes are involved in cancer is in the repression of retinoic acidreceptor signalling.

The clear involvement of HDACs in the control of cell proliferation anddifferentiation suggests that aberrant HDAC activity may play a role incancer. The most direct demonstration that deacetylases contribute tocancer development comes from the analysis of different acutepromyelocytic leukemias (APL). In most APL patients, a translocation ofchromosomes 15 and 17 (t(15;17)) results in the expression of a fusionprotein containing the N-terminal portion of PML gene product linked tomost of RARα (retinoic acid receptor). In some cases, a differenttranslocation (t(11;17)) causes the fusion between the zinc fingerprotein PLZF and RARα. In the absence of ligand, the wild type RARαrepresses target genes by tethering HDAC repressor complexes to thepromoter DNA. During normal hematopoiesis, retinoic acid (RA) binds RARαand displaces the repressor complex, allowing expression of genesimplicated in myeloid differentiation. The RARα fusion proteinsoccurring in APL patients are no longer responsive to physiologicallevels of RA and they interfere with the expression of the RA-induciblegenes that promote myeloid differentiation. This results in a clonalexpansion of promyelocytic cells and development of leukaemia. In vitroexperiments have shown that TSA is capable of restoringRA-responsiveness to the fusion RARα proteins and of allowing myeloiddifferentiation. These results establish a link between HDACs andoncogenesis and suggest that HDACs are potential targets forpharmaceutical intervention in APL patients. (See, for example, Kitamuraet al., 2000; David et al., 1998; Lin et al., 1998).

Furthermore, different lines of evidence suggest that HDACs may beimportant therapeutic targets in other types of cancer. Cell linesderived from many different cancers (prostate, colorectal, breast,neuronal, hepatic) are induced to differentiate by HDAC inhibitors(Yoshida and Horinouchi, 1999). A number of HDAC inhibitors have beenstudied in animal models of cancer. They reduce tumour growth andprolong the lifespan of mice bearing different types of transplantedtumours, including melanoma, leukaemia, colon, lung and gastriccarcinomas, etc. (Ueda et al., 1994; Kim et al., 1999).

Thus, although HDAC inhibitors (HDACi) are a promising new class ofanti-cancer drug, the molecular basis for their selectivegrowth-inhibitory activity on cancer cells is at present unclear.

PRAME was first identified as an antigen in human melanoma that triggerscytotoxic T cell-mediated anti-tumour immune responses (Ikeda et al,1997). PRAME is also over-expressed in a variety of other humanmalignancies, including acute and chronic leukemias, non-small-cell lungcarcinoma, head and neck cancer, renal carcinoma, and its expression isprognostic for a poor clinical outcome in breast cancer (Ikeda, 1997;van't Veer 2002; van Baren, 1998; Neumann 1998; Boon, 2003). However, nofunction for PRAME has been described to date.

The present invention demonstrates that PRAME expression inhibitsretinoic acid-induced differentiation, growth arrest and apoptosis andthat PRAME is, therefore, a dominant repressor, or negative regulator,of RAR signalling. The invention also shows that PRAME suppresses theHDACi-mediated activation of RAR signalling. These discoveries of afunction for PRAME have opened up a new avenue for the treatment ofcancer, via the suppression of PRAME, as well as enabling thestratification of subjects prior to treatment with known anti-cancertreatments, such as retinoids and HDACi's.

SUMMARY OF THE INVENTION

Accordingly, in its first aspect, the present invention provides amethod of treatment of a tumour which comprises administering to asubject in need of treatment an effective amount of an inhibitor ofPRAME, in combination with a second agent selected from the group of aninhibitor of HDAC (an HDACi) and a retinoid.

In a further aspect, the method provides an inhibitor of PRAME and asecond agent selected from the group of an inhibitor of HDAC (an HDACi)and a retinoid, as a combined preparation for simultaneous, separate orsequential use in therapy.

The invention also provides the use of an inhibitor of PRAME incombination with an HDACi or a retinoid for the manufacture of amedicament as a combined preparation for simultaneous, separate orsequential use in the treatment of a tumour.

The invention further provides the use of an HDACi, or a retinoid, fortreating a tumour in a subject, wherein the subject has receivedtreatment so as to suppress the level of PRAME in the tumour at the timeof HDACi or retinoid administration.

The invention further provides a methods of stratification of patientsbased on PRAME levels. This stratification may be used in diagnosis orprognosis for selection of patients for treatment, or to determine thelikely effectiveness of treatment.

The present invention also provides an assay for an inhibitor of aninteraction between PRAME and a retinoic acid receptor (RAR) whichcomprises bringing together:

-   -   (i) a candidate inhibitor; and    -   (ii) a PRAME protein and a RAR protein; and        determining if the putative inhibitor is capable of preventing        an interaction between said PRAME and RAR proteins.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: PRAME is a repressor of RAR signalling. a, 293 cells weretransfected with a GAL4-PRAME chimeric construct and a GAL4-luciferasereporter. b, 293 cells were transfected as in a, and were treated withPXD101. c, Ras^(V12) MEFs were transfected with a RA-responsiveluciferase reporter (RARE-luc) and either PRAME or empty vector, andtreated with RA. d, Ras^(V12) MEFs were transfected as in c, and treatedwith PXD101. e, Ras^(V12) MEFs were transfected with RARE-luciferase andeither RARα or empty vector, and were treated with PXD101.

FIG. 2: PRAME and its interaction with RAR. a, Schematic representationof PRAME. Amino acid residue numbers of the seven NR box motifs areindicated. b, B16 melanoma cells were transfected with RARE-luc andeither PRAME or PRAME-ΔLXXLL, and treated with RA. Average values are ofthree independent transfections (±s.d.).

FIG. 3: Effects of PRAME expression. Proliferation curve ofA375-PRAME^(KD) cells grown in normal medium or medium supplemented with5 μM RA.

FIG. 4: PRAME represses RAR signalling. a, B16 melanoma cells weretransfected with RARE-luciferase and either PRAME or empty vector andtreated with RA. b, Ras^(V12) MEFs were transfected as in a, and treatedwith TSA. c, B16 melanoma cells were transfected with a RARβ2-promoterluciferase reporter (RARβ2-luc) and either PRAME or empty vector andtreated with RA. d, Ras^(V12) MEFs were transfected with aRARβ2-luciferase reporter (R140-luc) or a RARβ2-luciferase reporter witha mutated RARE (M3M7-luc) and either PRAME or empty vector and treatedwith RA. Average values are of three independent transfections (±s.d.).

FIG. 5: Effects of PRAME expression on RA-signalling in melanoma. a, b,FM6 (a) and SK23 (b) human melanoma cells were transfected withRARE-luciferase and either pRS-PRAME or empty vector and treated withRA. c, A375 cells were transfected as in a, and treated with PXD101. d,B16 melanoma cells were transfected with a p21-promoter luciferaseconstruct and either PRAME or empty vector and treated with RA. Averagevalues are of three independent transfections (±s.d.)

DETAILED DESCRIPTION OF THE INVENTION

Inhibitor of PRAME

An inhibitor of PRAME includes any compound capable of either preventingexpression of PRAME or of preventing PRAME when expressed from exertingits normal activity. Normal activity, as used in this respect, refers toany activity performed by the PRAME expression product in the human oranimal body. Typically such activities include the repression oftranscription and/or interaction with RAR.

A preferred inhibitor of PRAME is an interfering RNA (RNAi). RNAinterference is the process of sequence-specific, post-transcriptionalgene silencing in animals and plants, initiated by double-stranded RNA(dsRNA) that is homologous in sequence to the silenced gene. RNAinterference (RNAi) is a process whereby the introduction of doublestranded RNA (dsRNA) into a cell inhibits gene expressionpost-translationally, in a sequence dependent fashion. This process isalso known as post-transcriptional gene silencing. Current models ofRNAi indicate that it is mediated by short (typically 20-25 nucleotides)dsRNAs known as ‘small interfering RNAs’ (siRNA). It appears that dsRNAis cleaved in the cell to create siRNAs. siRNAs are then incorporatedinto an RNA-induced silencing complex (RISC), guiding the complex to thehomologous endogenous mRNA. The activated RISC then cleaves the mRNAtranscript, resulting in the destruction of the mRNA in a cell which ishomologous to the siRNAs. The siRNAs are re-cycled. In this way, arelatively small number of siRNAs can selectively destroy a large excessof cellular mRNA.

To induce RNA interference in a cell, dsRNA may be introduced into thecell via a transgene, plasmid or virus which brings about expression ofthe siRNA in the cell. Alternatively, siRNA may be synthesised andintroduced directly into the cell, optionally in the form of apharmaceutical composition.

The complementary strands of the siRNA may be between 10 nucleotides(nt) and 30 nt in length, preferably between 20 nt and 25 nt.Preferably, the siRNA is 20, 21 or 22 nt in length. Generally, thenucleotides form a complementary double strand which may have shortoverhangs of one or two nt.

The siRNA sequence may be based on a contiguous sequence of 10-30nucleotides from the cDNA sequence of PRAME. The human cDNA sequence ofPRAME is available GenBank accession no. BC014074 and is shown herein asSEQ ID NO:1. It is known that certain residues of SEQ ID NO:1 are sitesof single nucleotide polymorphisms, namely residues 177 (T/C), 621(C/A), 924 (T/C), 1421 (T/C), 1685 (T/C) and 1966 (T/A) and thus siRNAsdesigned to target regions including any of these SNPs may include oneor other of the SNP residues, or the siRNA may be a mixture thereof.Preferably however the siRNA will be targeted to a region of thesequence which does not contain a known SNP.

The siRNA may be used to target a coding or non-coding region of SEQ IDNO:1. The coding region comprises 159 to 1688 of SEQ ID NO:1. Aparticular region of SEQ ID NO:1 which may be targeted is the 21nucleotide region of 714-734, shown below as SEQ ID NO:2.

siRNA molecules may be synthesized using standard solid or solutionphase synthesis techniques which are known in the art. Linkages betweennucleotides may be phosphodiester bonds or alternatives, for example,linking groups of the formula P(O)S, (thioate); P(S)S, (dithioate);P(O)NR′2; P(O)R′; P(O)OR6; CO; or CONR′2 wherein R is H (or a salt) oralkyl (1-12C) and R6 is alkyl (1-9C) is joined to adjacent nucleotidesthrough-O-or-S—.

Modified nucleotide bases can be used in addition to the naturallyoccurring bases, and may confer advantageous properties on siRNAmolecules containing them.

For example, modified bases may increase the stability of the siRNAmolecule, thereby reducing the amount required for silencing. Theprovision of modified bases may also provide siRNA molecules which aremore, or less, stable than unmodified siRNA.

The term ‘modified nucleotide base’ encompasses nucleotides with acovalently modified base and/or sugar. For example, modified nucleotidesinclude nucleotides having sugars which are covalently attached to lowmolecular weight organic groups other than a hydroxyl group at the3′position and other than a phosphate group at the 5′position. Thusmodified nucleotides may also include 2′substituted sugars such as2′-O-methyl-; 2-O-alkyl; 2-O-allyl; 2′-S-alkyl; 2′-S-allyl; 2′-fluoro-;2′-halo or 2; azido-ribose, carbocyclic sugar analogues a-anomericsugars; epimeric sugars such as arabinose, xyloses or lyxoses, pyranosesugars, furanose sugars, and sedoheptulose.

Modified nucleotides are known in the art and include alkylated purinesand pyrimidines, acylated purines and pyrimidines, and otherheterocycles. These classes of pyrimidines and purines are known in theart and include pseudoisocytosine, N4,N4-ethanocytosine,8-hydroxy-N6-methyladenine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil, 5 fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentyladenine, 1-methyladenine,1-methylpseudouracil, 1-methylguanine, 2,2-dimethylguanine,2methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine,N6-methyladenine, 7-methylguanine, 5-methylaminomethyl uracil, 5-methoxyamino methyl-2-thiouracil, 5-methoxycarbonylmethyluracil,5methoxyuracil, 2 methylthio-N6-isopentenyladenine, uracil-5-oxyaceticacid methyl ester, pseudouracil, 2-thiocytosine, 5-methyl-2 thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acidmethylester, uracil 5-oxyacetic acid, queosine, 2-thiocytosine,5-propyluracil, 5-propylcytosine, 5-ethyluracil, 5ethylcytosine,5-butyluracil, 5-pentyluracil, 5-pentylcytosine, 2,6,diaminopurine,methylpseudouracil, 1-methylguanine, 1-methylcytosine.

Alternatively, siRNA molecules or longer dsRNA molecules may be maderecombinantly by transcription of a nucleic acid sequence, preferablycontained within a vector as described below. Such a vector may be onedesigned to be introduced into a target tumour cell so as to produce ansiRNA in vivo. Where the siRNA is produced by recombinant means, the twostrands of the siRNA may be produced in separate transcripts. Moredesirably however the siRNA may be made in the form of a singletranscript which forms a hairpin loop structure comprising adouble-stranded siRNA sequence, for example as described in Brummelkampet al, Science 2002.

Thus it will be understood that an inhibitor of PRAME includes arecombinant vector capable of expressing an siRNA in a target tumourcell. Such a vector may comprise a sequence coding for the siRNAoperably linked to a promoter. The promoter may be any promoter suitablefor the transcription of the siRNA, such as a eukaryotic promoter. Sucha promoter may be an RNA gene promoter, such as a promoter whichproduces a small RNA transcript which preferably has a defined 3′ endlacking a polyA tail. A preferred such promoter is the polymerase-III H1gene RNA promoter.

The vector may be a viral vector, such as an adenovirus, herpes virus,vaccinia virus or retrovirus vector. Various viral vector systems fordelivery to a human or animal subject are known in the art, for exampleas described in U.S. Pat. Nos. 6,228,844 and 6,339,068, the contents ofwhich are incorporated herein by reference. The vector will include thesiRNA-encoding sequence operably linked to a promoter, as a constructcarried by the vector either in place of a native vector gene orinserted as additional DNA within the vector.

Further inhibitors of PRAME may be identified using the assay describedherein below which forms a further aspect of the present invention.

Inhibitor of HDAC

HDAC inhibitors suitable for use in the treatment of cancer are known inthe art. Typical HDACi include trichostatin A (TSA), trapoxin,suberoylanilide hydroxamic acid (SAHA), and phenylbutyrate, which havebeen reported to act, at least in part, by inhibiting histonedeacetylase (see, e.g., Yoshida et al., 1990; Richon et al., 1998;Kijima et al., 1993).

Additionally, diallyl sulfide and related molecules (see, e.g., Lea etal., 1999), oxamflatin (see, e.g., Kim et al., 1999; Sonoda et al.,1996), MS-27-275, a synthetic benzamide derivative (see, e.g., Saito etal., 1999; Suzuki et al., 1999; note that MS-27-275 was later re-namedas MS-275), butyrate derivatives (see, e.g., Lea and Tulsyan, 1995),FR901228 (see, e.g., Nokajima et al., 1998), depudecin (see, e.g., Kwonet al., 1998), and m-carboxycinnamic acid bishydroxamide (see, e.g.,Richon et al., 1998) have been reported to inhibit histone deacetylases.In vitro, some of these compounds are reported to inhibit the growth offibroblast cells by causing cell cycle arrest in the G1 and G2 phases,and can lead to the terminal differentiation and loss of transformingpotential of a variety of transformed cell lines (see, e.g., Richon etal, 1996; Kim et al., 1999; Yoshida et al., 1995; Yoshida & Beppu,1988). In vivo, phenylbutyrate is reported to be effective in thetreatment of acute promyelocytic leukemia in conjunction with retinoicacid (see, e.g., Warrell et al., 1998). SAHA is reported to be effectivein preventing the formation of mammary tumours in rats, and lung tumoursin mice (see, e.g., Desai et al., 1999).

A preferred class of inhibitors are those described in the followingpublications, the contents of which are incorporated herein byreference:

-   Watkins, C., et al., 2002, “Carbamic acid compounds comprising a    sulfonamide linkage as HDAC inhibitors,” published international    (PCT) patent application number WO 02/30879 (PCT/GB01/04326)    published 18 Apr. 2002;-   Watkins, C., et al., 2002, “Carbamic acid compounds comprising an    ether linkage as HDAC inhibitors,” published international (PCT)    patent application number WO 02/26703 (PCT/GB01/04327) published 4    Apr. 2002;-   Watkins, C., et al., 2002, “Carbamic acid compounds comprising an    amide linkage as HDAC inhibitors,” published international (PCT)    patent application number WO 02/26696 (PCT/GB01/04329) published 4    Apr. 2002; and-   Watkins, C., et al., 2003, “Carbamic acid compounds comprising a    piperazine linkage as HDAC inhibitors,” published international    (PCT) patent application number WO03/082288 (PCT/GB03/01463)    published 9 Oct. 2003.

A particularly preferred compound is known as PXD101(N-hydroxy-3-(3-phenylsulfamoyl-phenyl)-acrylamide), the structure ofwhich is:

This compound, or a pharmaceutically acceptable salt thereof, may beused in accordance with the present invention.

Retinoid

Compounds of retinoid type are compounds with a biological activityprofile similar to that of all-trans-retinoic acid or 9-cis-retinoicacid, which compounds themselves may be used in the practice of theinvention. These compounds can modify the expression of genes by meansof receptors of the retinoic acid family, such as the RARs and RXRS.Thus, retinoids may exhibit activity in the test of differentiation ofmouse embryonic teratocarcinoma cells (F9) (Strickland et al 1983)and/or in the test of inhibition of ornithine decarboxylase afterinduction with TPA in mice (Verma et al, 1978). These tests show theactivities of these compounds in the fields of cell differentiation andcell proliferation, respectively.

A wide variety of retinoids are known in the art. For example, US patentapplication 20030055110, the contents of which are incorporated hereinby reference, describes numerous retinoid compounds. The followingcompounds in particular were found to have activity of the RAR receptorantagonist type:4-[4-(6-Methoxymethoxy-4′-methylbiphenyl-2-yl)but-3-en-1-ynyl]benzo-icacid; 4-[4-(6-Methoxy-4′-methylbiphenyl-2-yl)but-3-en-1-ynyl]benzoicacid; 4-(6-Methoxyethoxymethoxy-7-(1-adamantyl)-2-naphthyl]salicylicacid;(E)-4-[4-(5-Methoxymethoxy-4′-methylbiphenyl-2-yl)but-3-en-1-ynyl]benzoicacid; 4-[4-(3-Methoxy-4′-methylbiphenyl-2-yl)but-3-en-1-ynyl]benzoicacid;2-Methoxymethoxy-2′-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthal-en-2-yl)[1,1,4′1″]terphenyl-4″-carboxylicacid; 4-[4-(4′-Methylbiphenyl-2-yl)but-3-en-1-ynyl]benzoic acid; and4-(4-(4′-Propylbiphenyl-2-yl)but-3-en-1-ynyl]benzoic acid.

Retinoid compounds such as these and others which exhibit activity atthe RAR receptor (by which is meant causing the receptor to induceexpression of a RA-responsive gene, for example the RARβ gene asdescribed herein), may be suitable for use in the present invention.

Treatment of a Tumour

The present invention may be applied to the treatment of a wide varietyof tumours, particularly those which are associated with over-expressionof HDAC, for example as a result of gene amplification or loss ofregulation of the HDAC gene.

Tumours in which such over-expression may occur include melanomas, acuteand chronic leukemias, non-small-cell lung carcinoma, head and neckcancer, renal carcinoma, and breast cancer.

By “treatment”, it will be understood that this includes anyintervention designed to alleviate the condition of the patient, e.g. byslowing down the rate of tumour progression, by providing an adjunct toother tumour therapies including surgery, by stabilizing the tumour orachieving partial or complete remission of the tumour.

Administration

The present findings indicate that the use of a PRAME inhibitor (PRAMEi)will allow HDAC inhibitors or retinoids to be used more effectively,where the PRAME inhibitor is administered with the HDACi or retinoid.Where reference is made to the administration of an HDACi or a retinoid,the invention also includes the administration of both.

By “simultaneous” administration, it is meant that the PRAMEi and theHDACi (or retinoid) are administered to a subject in a single dose bythe same route of administration.

By “separate” administration, it is meant that the PRAMEi and the HDACi(or retinoid) are administered to a subject by two different routes ofadministration which occur at the same time. This may occur for examplewhere one agent is administered by infusion and the other is givenorally during the course of the infusion.

By “sequential” it is meant that the two agents are administered atdifferent points in time, provided that the activity of the firstadministered agent is present and ongoing in the subject at the time thesecond agent is administered. For example, the PRAMEi may beadministered first, such that the amount of PRAME protein in the tumourcells of the subject is reduced at the point in time when the HDACiand/or retinoid is administered.

Generally, a sequential dose will occur such that the second of the twoagents is administered within 48 hours, preferably within 24 hours, suchas within 12, 6, 4, 2 or 1 hour(s) of the first agent.

The agents will be formulated appropriately for their desired route ofadministration. The agent or pharmaceutical composition comprising theagent may be administered to a subject by any convenient route ofadministration, whether systemically/peripherally or topically (i.e., atthe site of desired action).

Routes of administration include, but are not limited to, oral (e.g, byingestion); buccal; sublingual; transdermal (including, e.g., by apatch, plaster, etc.); transmucosal (including, e.g., by a patch,plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., byeyedrops); pulmonary (e.g., by inhalation or insufflation therapy using,e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., bysuppository or enema); vaginal (e.g., by pessary); parenteral, forexample, by injection, including subcutaneous, intradermal,intramuscular, intravenous, intraarterial, intracardiac, intrathecal,intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal,intratracheal, subcuticular, intraarticular, subarachnoid, andintrasternal; by implant of a depot or reservoir, for example,subcutaneously or intramuscularly.

The amount of a PRAMEi, HDACi and retinoid to be administered willultimately be at the discretion of the physician taking into account theage and status of the subject, and the particular activity of the agentbeing administered. Administration in vivo can be effected in one dose,continuously or intermittently throughout the course of treatment.Methods of determining the most effective means and dosage ofadministration are well known to those of skill in the art and will varywith the formulation used for therapy, the purpose of the therapy, thetarget cell being treated, and the subject being treated. Single ormultiple administrations can be carried out with the dose level andpattern being selected by the treating physician.

For example, where the PRAME is an siRNA, doses of siRNA may be in therange of from 0.001 μg to 100 g mg/kg body weight per dose. Doses may beadministered daily or at other intervals determined by the physician.Where the siRNA is delivered to the site of a tumour in the form of avector, the dose may be in the range of from 10⁵ to 10⁸ copies of thevector, which where the vector is in the form of a viral vector, mayequate to the number of viral particles. For HDACi compounds the dosemay be in the range of from 0.1 to about 250 mg per kilogram body weightof the subject per day. Retinoids may be administered in the range ofdosages of the order of from 0.05 to 10 mg/kg/day, preferably 0.1 to 7.5mg/kg/day, more preferably 0.1 to 2 mg/kg/day, administered once or, individed doses, 2 to 4 times per day.

Administration to Prior-treated Subject.

In a particular aspect, the invention relates to the use of an HDACi ora retinoid for the treatment of a novel subject type, namely a subjectin whom the level of expression of PRAME in a tumour present in saidsubject has been suppressed by medical treatment at the time of HDACi orretinoid treatment.

The suppression of expression of PRAME may have been achieved by use ofa PRAMEi as described herein. The patient will be one in which thePRAMEi has been administered recently enough so as to enhance theefficacy of the HDACi or retinoid, compared to the efficacy without theprior treatment. Desirably, the patient will have been treated with thePRAMEi within the previous 48, preferably 24, more preferably 12, suchas 6, 4, 2 or 1 hour(s)

In one embodiment, the patient may be characterised as a patient inwhich PRAME siRNA is present in the body of the patient.

Stratification and Selection of Subjects

The present findings also allow the stratification and/or selection ofsubjects for treatment with a HDACI or retinoid.

In this aspect of the invention, it will be necessary to first establishor obtain data from a cohort (e.g. at least 20) existing tumour patientsto determine the level of PRAME expression in their tumour. The level ofexpression may be determined at the level of RNA expression, e.g. usingRT-PCR, or at the level of protein expression, e.g. using anantibody-based approach. Such methods are known per se in the art. Theexpression levels will be distributed between low, intermediate and highvalues. It will be appreciated that what is determined to be of a low,intermediate and high value will be to some extent an arbitrarydesignation depending upon the criteria applied by any one particulartreatment centre, in a similar manner to, for example, biochemicalmarkers used in prenatal diagnoses. However this does not prevent themethod being practiced to the extent that the levels of PRAME can bedetermined in new subjects and compared to the collected data toestablish predictions or dosings in accordance with the invention as setout herein below.

In one aspect, the present invention may be used to predict theeffectiveness of a course of treatment with HDACi or a retinoid, or toselect patient in which treatment with HDACi or a retinoid is morelikely to be effective.

Accordingly in one aspect the invention provides a method comprising:

-   -   determining the level of expression of PRAME in a tumour of a        subject;    -   comparing said level to the levels previously determined in a        cohort of patients; and    -   treating said patient with a HDACi or a retinoid if said level        is indicative of low expression.

It will be understood that the step of comparing may be performed onhistoric data, and that it is not necessary to repeat the determinationfor that cohort each time the above method is practiced.

By “low expression” it is preferably meant a level in the lowerone-third, preferably the lower quartile of the distribution of thecohort.

The cohort to which reference is made is desirably matched for one ormore of tumour type, age, sex or severity of disease.

In another aspect the invention provides a method comprising:

-   -   determining the level of expression of PRAME in a tumour of a        subject;    -   comparing said level to the levels previously determined in a        cohort of patients; and    -   treating said patient with a PRAMEi and one or both of an HDACi        or a retinoid if said level is indicative of high expression.

By “high expression” it is preferably meant a level in the upperone-third, preferably the upper quartile of the distribution of thecohort.

In a similar manner, the invention may also be used to determine thefrequency or amount of HDACi or retinoid administered to a patient, withmore frequent or higher doses being administered to subjects withmoderate to high levels of PRAME expression.

Assay Methods

In another aspect, the finding that PRAME interacts directly with theRAR provides a novel target for agents capable of modulating the growth,differentiation and vitality of cells.

Accordingly, candidate inhibitors of the interaction between PRAME andRAR are identified by a method comprising bringing together:

-   a candidate inhibitor; and-   a PRAME protein and a RAR protein; and-   determining if the candidate inhibitor is capable of preventing an    interaction between said PRAME and RAR proteins.

For the purposes of this aspect of the invention, a PRAME protein willcomprise a eukaryotic, preferably mammalian, for example a rodent (e.g.murine) or primate, e.g. human PRAME protein or a fragment thereofcapable of forming a complex with a full length wild-type RAR protein,particularly human RAR.

Human PRAME protein is encoded by SEQ ID NO:1 shown herein. Proteinsfrom other species may also be used, and obtained for example byhomology searches of databases of genome sequences where such areavailable, or by recombinant molecular DNA techniques such as utilizingall or part of SEQ ID NO:1 as a probe against a genomic or cDNA libraryfrom a species of interest.

Fragments of PRAME will preferably comprise at least one of the seven“LXXLL” sequences identified herein, such as at least 2, 3, 4, 5 or 6such sequences. Fragments nay be at least 100, such as at least 200,e.g. at least 300, for example at least 400 amino acids in size.Fragments of these preferred sizes will desirably contain theabove-mentioned preferred numbers of LXXLL motifs.

For the purposes of this aspect of the invention, a retinoic acidreceptor alpha (RAR) protein will comprise a eukaryotic, preferablymammalian, for example a rodent (e.g. murine) or primate, e.g. human RARprotein or a fragment thereof capable of forming a complex with a fulllength wild-type PRAME protein, particularly human PRAME. The proteinmay be the alpha 1 or alpha 2 isoform.

A number of different RAR alpha proteins are available on publicdatabases. Human RAR-alpha is SwissProt accession P10276, and murine isSwissProt P11416. The rat alpha 2 isoform is Genbank accessionAAC23439.1. Non-mammalian versions of the protein have beencharacterised in chick (SwissProt Q90966), Xenopus (SwissProt P51126)and pufferfish (SwissProt Q9W5Z3).

Fragments may be at least 100, such as at least 200, e.g. at least 300,for example at least 400 amino acids in size.

Assays according to the invention may be performed in any formatavailable to the person skilled in the art. The precise format of theassay of the invention may be varied by those of skill in the art usingroutine skill and knowledge.

For example, the interaction between a PRAME protein and an RAR may bestudied by labelling one with a detectable label and bringing it intocontact with the other which has been immobilised on a solid support.Suitable detectable labels include ³⁵S-methionine which may beincorporated into recombinantly produced PRAME and/or RAR. Therecombinantly produced PRAME and/or RAR may also be expressed as afusion protein containing an epitope which can be labelled with anantibody.

The protein which is immobilized on a solid support may be immobilizedusing an antibody against that protein bound to a solid support or viaother technologies which are known per se. A preferred in vitrointeraction may utilise a fusion protein includingglutathione-S-transferase (GST). This may be immobilized on glutathioneagarose beads. In an in vitro assay format of the type described abovethe putative modulator compound can be assayed by determining itsability to modulate the amount of labelled PRAME or RAR which binds tothe immobilized GST-RAR or GST-PRAME, as the case may be. This may bedetermined by fractionating the glutathione-agarose beads bySDS-polyacrylamide gel electrophoresis. Alternatively, the beads may berinsed to remove unbound protein and the amount of protein which hasbound can be determined by counting the amount of label present in, forexample, a suitable scintillation counter.

Alternatively an antibody attached to a solid support and directedagainst one of PRAME or RAR may be used in place of GST to attach themolecule to the solid support. Antibodies against PRAME and RAR may beobtained in a variety of ways known as such in the art, and as discussedherein.

In an alternative mode, one of PRAME and RAR may be labelled with afluorescent donor moiety and the other labelled with an acceptor whichis capable of reducing the emission from the donor. This allows an assayaccording to the invention to be conducted by fluorescence resonanceenergy transfer (FRET). In this mode, the fluorescence signal of thedonor will be altered when PRAME and RAR interact. The presence to acandidate modulator compound which modulates the interaction willincrease the amount of unaltered fluorescence signal of the donor.

FRET is a technique known per se in the art and thus the precise donorand acceptor molecules and the means by which they are linked to PRAMEand RAR may be accomplished by reference to the literature.

Suitable fluorescent donor moieties are those capable of transferringfluorogenic energy to another fluorogenic molecule or part of a compoundand include, but are not limited to, coumarins and related dyes such asfluoresceins, rhodols and rhodamines, resorufins, cyanine dyes, bimanes,acridines, isoindoles, dansyl dyes, aminophthalic hydrazines such asluminol and isoluminol derivatives, aminophthalimides,aminonaphthalimides, aminobenzofurans, aminoquinolines,dicyanohydroquinones, and europium and terbium complexes and relatedcompounds.

Suitable acceptors include, but are not limited to, coumarins andrelated fluorophores, xanthenes such as fluoresceins, rhodols andrhodamines, resorufins, cyanines, difluoroboradiazaindacenes, andphthalocyanines.

A preferred donor is fluorescein and preferred acceptors includerhodamine and carbocyanine. The isothiocyanate derivatives of thesefluorescein and rhodamine, available from Aldrich Chemical Company Ltd,Gillingham, Dorset, UK, may be used to label PRAME and RAR. Forattachment of carbocyanine, see for example Guo et al, J. Biol. Chem.,270; 27562-8, 1995.

The above assay formats may also be used to determine the ability of aputative modulator compound to modulate the interaction of PRAME withRAR. Such assays are optionally performed in the presence of a retinoidcompound, such as retinoic acid.

Assays of the invention may also be performed in vivo. Such an assay maybe performed in any suitable host cell, e.g a bacterial, yeast, insector mammalian host cell. Yeast and mammalian host cells are particularlysuitable.

To perform such an assay in vivo, constructs capable of expressing PRAMEand RAR and a reporter gene construct may be introduced into the cells.This may be accomplished by any suitable technique, for example calciumphosphate precipitation or electroporation. The three constructs may beexpressed transiently or as stable episomes, or integrated into thegenome of the host cell.

In vivo assays may also take the form of two-hybrid assays wherein PRAMEand RAR are expressed as fusion proteins, one being a fusion proteincomprising a DNA binding domain (DBD), such as the yeast GAL4 bindingdomain, and the other being a fusion protein comprising an activationdomain, such as that from GAL4 or VP16. In such a case the host cell(which again may be bacterial, yeast, insect or mammalian, particularlyyeast or mammalian) will carry a reporter gene construct with a promotercomprising a DNA binding elements compatible with the DBD. The reportergene may be a reporter gene as disclosed above. The promoters for thegenes may be those discussed above.

PRAME and RAR and the reporter gene, may be introduced into the cell andexpressed transiently or stably.

Candidate inhibitor compounds may be natural or synthetic chemicalcompounds used in drug screening programmes. Extracts of plants,microbes or other organisms, which contain several characterised oruncharacterised components, may also be used. Combinatorial librarytechnology (including solid phase synthesis and parallel synthesismethodologies) provides an efficient way of testing a potentially vastnumber of different substances for the ability to modulate aninteraction. Such libraries and their use are known in the art for allmanner of natural products, small molecules and peptides, among others.Many such libraries are commercially available and sold for drugscreening programmes of the type now envisaged by the present invention.

A further class of candidate inhibitor comprises antibodies or bindingfragments thereof which bind a protein target.

Examples of antibody fragments, capable of binding an antigen or otherbinding partner, are the Fab fragment consisting of the VL, VH, Cl andCH1 domains; the Fd fragment consisting of the VH and CH1 domains; theFv fragment consisting of the VL and VH domains of a single arm of anantibody; the dAb fragment which consists of a VH domain; isolated CDRregions and F(ab′)2 fragments, a bivalent fragment including two Fabfragments linked by a disulphide bridge at the hinge region. Singlechain Fv fragments are also included. An antibody specific for a proteinmay be obtained from a recombinantly produced library of expressedimmunoglobulin variable domains, e.g. using lambda bacteriophage orfilamentous bacteriophage which display functional immunoglobulinbinding domains on their surfaces; for instance see WO92/01047. Such atechnique allows the rapid production of antibodies against an antigen,and these antibodies may then be screening in accordance with theinvention.

Another class of candidate molecules is peptides, based upon a fragmentof the protein sequence to be inhibited. In particular, fragments of theprotein corresponding to portions of the protein which interact withother proteins or with DNA, may be a target for small peptides which actas competitive inhibitors of protein function. Such peptides may be forexample from 5 to 20 amino acids in length.

The peptides may also provide the basis for design of mimetics. Suchmimetics will be based upon analysis of the peptide to determine theamino acid residues or portions of their side chains essential andimportant for biological activity to define a pharmacophore followed bymodelling of the pharmacophore to design mimetics which retain theessential residues or portions thereof in an appropriatethree-dimensional relationship. Various computer-aided techniques existin the art in order to facilitate the design of such mimetics.

Inhibitors obtained in accordance with these aspects of the presentinvention may be used in methods of treating a cancer in a subject.

The present invention therefore also provides a pharmaceuticalcomposition comprising an inhibitor of the interaction between PRAME andRAR together with a pharmaceutically acceptable carrier therefor.

Generally, the inhibitor will be formulated with one or morepharmaceutically acceptable carriers suitable for a chosen route ofadministration to a subject. For solid compositions, conventionalnon-toxic solid carriers include, for example, pharmaceutical grades ofmannitol, lactose, cellulose, cellulose derivatives, starch, magnesiumstearate, sodium saccharin, talcum, glucose, sucrose, magnesiumcarbonate, and the like may be used. Liquid pharmaceuticallyadministrable compositions can, for example, be prepared by dissolving,dispersing, etc, a inhibitor and optional pharmaceutical adjuvants in acarrier, such as, for example, water, saline aqueous dextrose, glycerol,ethanol, and the like, to thereby form a solution or suspension. Ifdesired, the pharmaceutical composition to be administered may alsocontain minor amounts of non-toxic auxiliary substances such as wettingor emulsifying agents, pH buffering agents and the like, for example,sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate,sorbitan monolaurate, triethanolamine oleate, etc. Actual methods ofpreparing such dosage forms are known, or will be apparent, to thoseskilled in this art; for example, see Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975. Thecomposition or formulation to be administered will, in any event,contain a quantity of the active compound(s) in an amount effective toalleviate the symptoms of the subject being treated.

Routes of administration may depend upon the precise condition beingtreated, though since endothelial cells form the lining of thevasculature, administration into the blood stream (e.g. by i.v.injection) is one possible route.

In addition, the present invention provides an inhibitor of theinteraction between PRAME and RAR for use in therapy.

Furthermore, the present invention provides the use of an inhibitor ofthe interaction between PRAME and RAR in the manufacture of a medicamentfor the treatment of a cancer.

Methods and Experimental

In order to address the molecular basis for the selectivegrowth-inhibitory activity of HDACi on cancer cells, we performed afunctional genetic screen to identify genes that confer resistance to asmall-molecule HDACi, named PXD101 (Plumb et al, 2003). OncogenicRAS^(V12)-transformed mouse embryonic fibroblasts (RAS^(V12)-MEFs) wereinfected with a high complexity retroviral cDNA expression libraryderived from human K562 erythroleukemia cells and exposed to 1 μM PXD101for three weeks. HDACi-resistant colonies appeared at low frequency onlyafter infection with cDNA expression library. Integrated proviruses weremobilized from PXD101-resistant colonies by Moloney virussuper-infection as described (Jacobs et al, 2000). After second roundselection, cDNA inserts were recovered by PCR and identified by DNAsequence analysis. Using this approach, we identified full-length cDNAsencoding the human melanoma tumour antigen PRAME (PReferentiallyexpressed Antigen in MElanoma (Ikeda et al, 1997) and Retinoic AcidReceptor alpha (RARα) as cDNAs that allow continuous proliferation inthe presence of 1 μM PXD101. PRAME also rescues these cells from growtharrest by a related HDACi, Trichostatin A at a concentration of 0.1 μM.

PRAME expression by retroviral infection of Ras^(V12) MEFs was comparedto endogenous PRAME expression levels in the human melanoma cell linesFM6, SK23, 453A0, A375, FM3 and D10. Cell extracts were immunoblottedfor PRAME and CDK4 (as a control). The level of PRAME required to conferresistance to HDACi in the RAS^(V12)-MEFs is comparable to that seen inseveral human melanoma cell lines.

Ras^(V12) MEFs were also transduced with PRAME or RARα retrovirus andtreated with 1 μM PXD101 for 16 hrs. Extracts were immunoblotted foracetyl-H3, p21, PRAME, RARα and CDK4 (control). It was found thatexpression of PRAME or RARα in RAS^(V12)-MEFs did not prevent theincrease in histone H3 acetylation nor the well-documented induction ofp21^(cip1) expression by HDACi-treatment(Richon et al 2000), suggestingthat PRAME and RARα act downstream of HDACs to suppress the effects ofthe HDACi on cell proliferation.

PRAME was first identified as an antigen in human melanoma that triggerscytotoxic T cell-mediated anti tumour immune responses (Ikeda et al1997). PRAME is also over-expressed in a variety of other humanmalignancies, including acute and chronic leukemias, non-small-cell lungcarcinoma, head and neck cancer, renal carcinoma, and its expression isprognostic for a poor clinical outcome in breast cancer (Ikeda et al1997; van 't Veer et al, 2002; van Baren et al, 1998; Nemann et al,1998, Boon et al, 2003). However, no function for PRAME has beendescribed to date. Since HDAC inhibitors affect gene transcription, weasked if PRAME acts as a repressor or activator of transcription. Totest this, we co-transfected a vector in which PRAME is fused to the DNAbinding domain of the yeast transcription factor GAL4 (GAL4-PRAME) witha GAL4-luciferase reporter plasmid in human 293 cells. FIG. 1 a showsthat expression of GAL4-PRAME caused a strong and dose-dependentinhibition of gene expression, suggesting that PRAME is a repressor oftranscription. Treatment of cells with HDACi did not affect this,indicating that PRAME repression is mostly HDAC-independent (FIG. 1 b).

Because we identified both PRAME and RARα in the genetic screendescribed above, we asked if these two proteins act in the same pathway.To address this, we asked if PRAME affects RAR signalling. Wetransfected PRAME into RAS^(V12)-MEFs together with a reporter genedriven by a Retinoic Acid Responsive Element (RARE-luciferase). FIG. 1 cshows that PRAME expression strongly inhibited RA-induced activation ofthe RA-responsive reporter gene over a wide range of RA concentrations.Similar results were obtained in mouse B16 melanoma cells (FIG. 4).PRAME also repressed RAR signalling induced by HDACi treatment (FIG. 1 dand FIG. 4). Importantly, ectopic expression of RARE also repressed theactivation of the RA-responsive reporter gene construct by HDACi (FIG. 1e). Thus, both cDNAs that we identified in the genetic screen describedabove suppress HDACi-mediated activation of the RA-responsive reportergene. This raises the possibility that suppression of HDACi-inducedRA-signalling (Lin et al 1998, and FIG. 1 c) is one way in which cellscan become resistant to HDACi treatment. However, our data do notexclude that RARα and PRAME act on other pathways to mediate escape fromHDACi-induced growth arrest.

To further address the effect of PRAME on RA-signalling we studied RARβ,as this gene contains a RA-responsive element in its promoter (de The etal 1990). To ask if PRAME expression affected the expression of theendogenous RARβ gene, F9 mouse embryo carcinoma cells were transfectedwith PRAME or empty vector, treated with 10⁻⁷ M RA, and immunoblottedfor RARβ. Average values of three independent transfections weredetermined. It was found that endogenous RARβ protein expression wasinduced only in parental and mock-transfected F9 cells, but not inPRAME-transfected F9 derivatives. PRAME also repressed RA-inducedactivation of a RARβ2 promoter-luciferase construct in bothRAS^(V12)-MEFs and B16 melanoma cells (FIG. 4). Together, these dataindicate that PRAME is a negative regulator of RA-signalling.

The observed effect of PRAME on RAR signalling could result from aphysical interaction of PRAME with RAR or from more indirect effects. Toask if PRAME physically interacts with RAR, we stably expressed aTAP-tagged (Rigaut et al, 1999) PRAME in RAS^(V12)-MEFs. Ras^(V12) MEFswere infected with TAP-PRAME, RARα, or GFP (control) retrovirus andcultured in the presence of 1 μM PXD101. TAP-PRAME was functional as itrescued cells from PXD101-induced growth arrest.

Ras^(V12) MEFs (MEF) were transduced with TAP-tagged PRAME andexpression levels were compared to endogenous PRAME in SK23 and A375melanoma. Importantly, TAP-PRAME protein levels were comparable to thelevels of endogenous PRAME in the human melanoma cell lines SK23 andA375.

TAP-PRAME was immunoprecipitated (using IgG beads (Rigaut et al, 1999),indicated as anti-TAP) before and after treatment with 1 μM RA andprecipitates were immunoblotted for RARα. It was found that endogenousRARα co-precipitates with TAP-PRAME, both in the absence and presence ofRA, indicating that PRAME and RARα form a ligand-independent complex atprotein concentrations seen in human tumours.

The amino-acid sequence of PRAME contains seven potential leucine-richnuclear receptor (NR) boxes (LXXLL motifs (Heery et al, 1997), FIG. 2c). Many modulators of nuclear receptor activity interact directly withtheir target receptors via one or more of these motifs (Heery et al,1997; Torchia et al, 1997). To test if these motifs in FRAME arerequired for inhibition of RAR signalling, we introduced point mutationsin PRAME, to change several of the leucine residues in each of the sevenNR boxes into valines. The resulting mutant, PRAME-ΔLXXLL, was expressedat comparable levels as wild type PRAME (as determined byimmunoblotting), but failed to repress RAR signalling (FIG. 2 b). Themutant did not bind RARα, when immunoprecipitations as described abovewere performed. We conclude that the LXXLL motifs in PRAME are requiredfor modulation of RAR activity.

RA induces proliferation arrest, differentiation and apoptosis in manycell types. We therefore asked if PRAME expression affected theRA-induced differentiation of F9 mouse embryo carcinoma cells towardsparietal endoderm (Strickland and Mahdavi, 1978). F9 cells were stablytransfected with PRAME or control vector and individual colonies wereselected. In the absence of RA, the morphology of all transfected cellswas the same as that of parental F9 cells. However, F9 cells stablytransfected with PRAME or empty vector and treated with 10⁻⁷ M RA wereresistant to RA-induced morphological differentiation and growth arrest.Apart from differentiating, a fraction of F9 cells treated with RA diesby apoptosis (Atencia et al, 1994). PRAME expression in F9 cells alsoconferred resistance to RA-induced apoptosis as cleaved caspase 3 wasapparent in vector controls, but not in PRAME transfectants. We concludethat PRAME expression confers resistance to RA-induced proliferationarrest, differentiation and apoptosis.

Human melanoma cells often have defects in RA signalling (Atencia et al,1994; van der Leede et al, 1993). As PRAME is over-expressed in some 90%of melanomas (Ikeda et al, 1997), we asked if PRAME expression explainedtheir RA-unresponsiveness. To test this, we inhibited PRAME expressionin several RA-resistant melanoma cell lines (Demary et al, 2001) throughRNA interference. We selected a unique 21-mer sequence in the PRAMEtranscript for cloning into pRETRO-SUPER (pRS), a vector that mediatessuppression of gene expression through synthesis of short hairpin RNAshaving small interfering RNA (siRNA)-like properties (Brummelkamp et al,2002a; Brummelkamp et al 2002b). 293 cells were transfected withflag-tagged PRAME and pRS-PRAME or pRS empty vector. Extracts wereimmunoblotted for flag or GFP (control). In addition, A375 cells weretransfected with RARE-luciferase and either pRS-PRAME or empty vectorand treated with RA. Average values of three independent transfectionswere determined. A375-PRAME^(KD) cells and control A375 cells wereimmunoblotted for PRAME, RARα, p21, and RARβ.

It was found that pRS-PRAME mediates effective decrease of ectopicallyexpressed PRAME as well as endogenous PRAME protein. To ask if knockdownof FRAME restores RA-responsiveness, we co-transfected A375 humanmelanoma cells, which express high levels of endogenous PRAME, withpRS-PRAME and a RA-responsive reporter gene construct. The PRAMEknockdown greatly enhanced RA-signalling in the A375 cells as determinedby using the RARE-luciferase construct. Similar results were found inthe SK23 and FM6 human melanoma cell lines (FIG. 5). Melanomas arerelatively resistant to treatment with HDACi (Plumb et al, 2003), butA375 cells transfected with PRS-PRAME had enhanced PXD101-induced RARsignalling compared to vector controls (FIG. 5).

To assess the effect of PRAME knockdown on cell proliferation, wegenerated stable derivatives of A375 melanoma having shRNA-mediatedknockdown of PRAME expression (A375-PRAME^(KD)). These A375-PRAME^(KD)cells were cultured for 15 days according to the 3T3 protocol. FIG. 3shows that PRAME^(KD) significantly decreased proliferation rates,especially when cultured in the presence of RA. Consistent with thenotion that PRAME^(KD) restores RA signalling, we found that the knownRA target genes RARβ and p21^(CIP1) (de The et al, 1990, Liu et al 1996)were significantly up-regulated in PRAME^(KD) cells, and their inductionwas further enhanced by 5 μM RA treatment. Consistent with this, a p21promoter-luciferase reporter was activated by RA in B16 melanoma cells,but its activity was strongly suppressed by PRAME (FIG. 5). RARα isdegraded by the proteasome in response to RA signalling (Zhu et al,1999) and in agreement with this, RARα protein levels were significantlydecreased in PRAME^(KD) cells. Together, these data indicate that PRAMEis a major regulator of RA-signalling, which contributes toRA-unresponsiveness of human melanomas.

The presence of NR boxes in PRAME (FIG. 2 a) suggests that theinteraction between PRAME and RARα takes place via one or more or moreof these motifs. To test if the NR boxes of PRAME are required forbinding to RAR and inhibition of RAR signaling, point mutations wereintroduced in each of the seven LXXLL motifs in FRAME by changingconserved leucine (L) residues into valines (V). The resulting PRAMEmutants were named after the respective NR boxes that were mutated. Oneadditional mutant was made in which all seven LXXLL motifs were mutated,referred to as PRAME-ΔLXXLL. MEFs were transfected with RARE-luc andPRAME or PRAME NR box mutants and treated with RA.

Six out of seven PRAME single NR box mutants inhibited RAR signaling toa similar extent as wild-type PRAME except for the box 7 PRAME-LREVVmutant. This mutant was as defective in repressing RAR signaling as thePRAME mutant in which all 7 NR boxes were mutated. Consistent with thisobservation, endogenous RARα failed to co-immunoprecipitate with aTAP-PRAME-LREVV mutant protein. To investigate if the repressionfunction of PRAME was affected by the LREVV mutation, we tested theeffect of cotransfection of a Gal4-PRAME-LREVV fusion protein onexpression of the Gal4-luciferase reporter. Gal4-PRAME-LREVV inhibitedexpression to a similar extent as Gal4-PRAME, indicating that repressionwas not affected.

A mammalian two-hybrid assay was performed in which a wild-type PRAMEconstruct, Gal4-PRAME-LRELL (416-509) or the box 7 mutantGal4-PRAMELREVV (416-509) was co-expressed with VP16-RARα LBD in thepresence of RA. Normalized luciferase activities were determined as theaverage of three independent transfections. The PRAME wildtype LRELL NRbox interacted with VP16-RARα, but introduction of the LREVV mutation inPRAME disrupted the association. Taken together, these data suggest thatan intact LRELL motif in PRAME is required for binding to RARα andrepression of RAR signaling.

Human melanomas often have defects in RAR signaling (Demary et al.,2001; van der Leede et al., 1993) and PRAME is over-expressed in 88% ofprimary melanomas and 95% of melanoma metastases (Ikeda et al., 1997).This raises the possibility that PRAME expression is responsible fortheir RA-unresponsiveness. To test this hypothesis, we inhibited PRAMEexpression in three different melanoma cell lines (A375, FM6 and SK23)through stable RNA interference (Brummelkamp et al., 2002a).Transfection of A375 human melanoma cells with a PRAME-specific shRNAvector (pRS-PRAME) caused a significant decrease in levels of endogenousPRAME. Knockdown of PRAME greatly enhanced RAR signaling in three humanmelanoma cell lines that are relatively insensitive to RA. Together,these data support the notion that PRAME expression confers cellularresistance to RA in human melanoma.

To examine the role of PRAME in RA-responsiveness in vivo, we used ahuman melanoma xenograft model. Nude mice were subcutaneouslytransplanted with parental A375 into one flank and A375-PRAMEKD cells inthe opposite flank and the mice were treated p.o. daily with either 5mg/kg RA or vehicle only, while tumor volumes were measured weekly.Tumor growth was severely retarded by RA treatment in PRAMEKD melanomas,but not in parental A375 melanomas that grew in a different anatomicallocation in the same mice. Together, these data suggest that PRAMEfunctions as a negative regulator of RAR signaling, which contributes toRA-unresponsiveness of human melanomas.

The data presented here indicate that PRAME is a dominant,ligand-independent, repressor of RAR signalling, resulting in adecreased cellular response to RA-driven differentiation, growth arrestand/or apoptosis. PRAME is distinct from the known co-repressors ofnuclear receptors such as N-CoR and SMRT, whose interaction with nuclearreceptors is lost upon ligand binding (Xu et al 1999). Our data alsoidentify PRAME expression as a novel mechanism by which tumour cells canescape tumour-suppressive RA signalling. In this respect, PRAMEexpression phenocopies the PML-RARα and PLZF-RARα translocations seen inacute promyelocytic leukemia. Hence, melanoma cells and other tumourcells that over-express PRAME have a selective advantage overPRAME-negative cells, which may explain why PRAME expression is retainedby tumour cells in vivo, despite the fact that its presence elicits acytotoxic T cell-mediated anti-tumour immune response (Ikeda et al.,1997). Finally, our data suggest a strategy to select subjects that arelikely to benefit most from treatment with HDACi compounds.

Methods

Reagents, Antibodies and Plasmid Construction

Full-length PRAME in pcDNA3.1/Neo(+) was a gift from Drs. M. Griffioenand C. Melief (Leiden, The Netherlands). Retroviral PRAME was generatedby cloning a BamHI-XhoI PRAME fragment into pMX-IRES-GFP (pMIG).TAP-tagged PRAME was generated by cloning a PCR amplified EcoRI PRAMEfragment into pZOME-1-N (Cellzome) and Gal4-PRAME was generated bycloning a BamHI-XbaI PRAME fragment into a GAL4-DBD expression vector.Retroviral RARα was generated by cloning an EcoRI digested RARα cDNAinto pMX. The PRAME-ΔLXXLL mutant was made using the QuikChangeSite-Mutagenesis kit (Stratagene). PRAME-ΔLXXLL has multiple of theleucines (L) in the LXXLL motifs changed into valines (V). The resultingsequences were: LDVVV, VRRLL, LDQVV, VQALL, LLAVV, LQSVV, LREVV.

pRS-PRAME was generated by ligating synthetic oligos against the targetsequence GGTGCCTGTGATGAATTGTTC (SEQ ID NO:2) into pRS in a mannersimilar to that described previously (Brummelkamp et al, 2002a). Thefollowing oligonucleotide sequences were annealed:

(SEQ ID NO:3) 5′-GATCCCCGGTGCCTGTGATGAATTGTTC TTCAAGAGA GAACAATTCATCACAGGCACCTTTTTGGAAA-3′ and (SEQ ID NO:4)5′-AGCTTTTCCAAAAAGGTGCCTGTGATGAATTGTTC TCTCTTGAAGAACAATTCATCACAGGCACCGGG-3′to provide a double strand with overhanging BamH1 ends. This was ligatedinto the vector. Expression from the H1-RNA promoter provides atranscript commencing at start of the target sequence (5′ underlinedregion in SEQ ID NO:3), through a hairpin loop (italics in SEQ ID NO:3above), and terminating at the end of the complement (3′ underlinedregion of SEQ ID NO:3) of the target sequence, with the addition of a 2nt overhang.

RA-responsive luciferase constructs and RARα cDNA were kindly providedby Dr. H. Stunnenberg (Nijmegen, The Netherlands). The erythroleukemiaretroviral cDNA library was provided by Dr. Koh and Dr. Daley(Cambridge, Mass.). All-trans-retinoic acid (ATRA) and TSA were fromSIGMA, and PXD101 was provided by Topotarget/Prolifix Ltd (Abingdon,UK). Anti-PRAME affinity-purified antibodies were a generous gift fromDr. P. Coulie (Brussels, Belgium) and were generated by immunizingrabbits with peptides FPEPEAAQPMTKKRKVDG (AH-151; SEQ ID NO:6) andCGDRTFYDPEPIL (AH-152; SEQ ID NO:7). Antibodies against RARα (C-20),RARβ (C-19), p21 (F5), GFP (FL), CDK4 (C-22), and CDK2 (H-298) were fromSanta Cruz Biotechnologies, anti-acetyl H3 was from Serotec, anti-Ras(R02120) was from Transduction laboratories, and anti-cleaved caspase-3(Asp 175) was from Cell Signaling.

Cell Cultures, Genetic Screen, and Colony Formation Assays

All cells were cultured in DMEM supplemented with 10% fetal calf serum(FCS). Phoenix packaging cells were used to generate ecotropicretroviruses as described (Serrano et al, 1997). p53^(−/−) MEFs wereinfected with pBabe-Puro-RAS^(V12) retrovirus and selected for puromycinresistance. The resulting RAS^(V12)-MEFs were infected with libraryretroviral supernatants and re-plated at a cell density of 5.10⁴cells/10 cm dish 48 hrs after infection. HDAC inhibitor PXD101 (1 μM) orTSA (0.1 μM) was added to the medium 16 hours after reseeding and themedium with HDAC inhibitor was refreshed every third day.

Transfections and Reporter Assays

Transfections were carried out using the Lipofectamine reagent(Invitrogen), except for Ras^(V12) MEFs and Phoenix cells which weretransfected using calcium phosphate precipitation. RA-based reporterassays were done in DMEM with charcoal-stripped FCS (Hyclone). Inreporter assays 0.5 μg of reporter-luciferase, 1 ng CMV-renilla, and 3μg of the indicated DNA were transfected. RA, PXD101, or TSA was added24 hrs after transfection and assays were performed 48 hrs aftertransfection. In knock-down experiments, RA or PXD101 was added 72 hrsafter transfection and assays were performed 96 hrs after transfection.Luciferase activities shown represent ratios between luciferase andrenilla internal control values.

Western Blotting and Co-imminoprecipitation

Cells were lysed in Ripa buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1%NP-40, 0.5% deoxycholic acid, and 0.1% SDS) supplemented with proteaseinhibitors (Complete; Roche) and 0.2 nM PMSF and proteins were separatedon 10-14% SDS-PAGE gels. Proteins were transferred to polyvinylidinedifluoride membranes (Immobilon P, Millipore) and Western blots wereprobed with the indicated antibodies. For TAP (co-)immunoprecipitationscells were lysed in ELB buffer (0.25 M NaCl, 0.1% NP-40, 50 mM HEPES pH7.3) supplemented with protease inhibitors and PMSF. Lysates wereincubated with IgG-coated sepharose beads (Amersham) toimmunoprecipitate TAP and TAP-PRAME (Rigaut et al 1999), indicated asanti-TAP, or with protein A beads and normal mouse serum (mock IP) for 2hrs, washed, and separated on SDS-PAGE gels.

Differentiation and Proliferation Assays

F9 cells were stably transfected with PRAME or empty vector andresistant cells were differentiated in 10⁻⁷ M RA for 5 days. A375 cellswere stably transfected with pRS-PRAME or empty vector and stable cloneswere cultured according to the 3T3 protocol. Medium with RA wasrefreshed every 24-48 hrs.

Mouse Tumor Xenografts

Female 5-6 week old athymic BALB-C nude mice (nu/nu) were s.c. implantedwith 1×10⁶ cells bilaterally into the axial regions. Each mouse receivedA375-PRAMEKD cells in its right flank and control A375 cells in its leftflank. Mice were randomized into treatment groups and treated with 5mg/kg RA or vehicle (ethanol in sunflower oil) p.o. with a 20-gaugeintragastric feeding tube daily. Tumor diameter was measured withcalipers weekly. The pRS vector which was used to generate A375-PRAMEKDcells is a self-inactivating retroviral vector, to prevent re-activationand spreading of virus (Brummelkamp et al., 2002a; Brummelkamp et al.,2002b). The experiment was performed twice, with n=20 and n=10 mice andresults were similar in both experiments.

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SEQ ID NO:1 - Human PRAME 1 gggaaaccga ctcctgggag cagggaggaa cgcgcgctccagagacaact tcgcggtgtg 61 gtgaactctc tgaggaaaaa cacgtgcgtg gcaacaagtgactgagacct agaaatccaa 121 gcgttggagg tcctgaggcc agcctaagtc gcttcaaaatggaacgaagg cgtttgtggg 181 gttccattca gagccgatac atcagcatga gtgtgtggacaagcccacgg agacttgtgg 241 agctggcagg gcagagcctg ctgaaggatg aggccctggccattgccgcc ctggagttgc 301 tgcccaggga gctcttcccg ccactcttca tggcagcctttgacgggaga cacagccaga 361 ccctgaaggc aatggtgcag gcctggccct tcacctgcctccctctggga gtgctgatga 421 agggacaaca tcttcacctg gagaccttca aagctgtgcttgatggactt gatgtgctcc 481 ttgcccagga ggttcgcccc aggaggtgga aacttcaagtgctggattta cggaagaact 541 ctcatcagga cttctggact gtatggtctg gaaacagggccagtctgtac tcatttccag 601 agccagaagc agctcagccc atgacaaaga agcgaaaagtagatggtttg agcacagagg 661 cagagcagcc cttcattcca gtagaggtgc tcgtagacctgttcctcaag gaaggtgcct 721 gtgatgaatt gttctcctac ctcattgaga aagtgaagcgaaagaaaaat gtactacgcc 781 tgtgctgtaa gaagctgaag atttttgcaa tgcccatgcaggatatcaag atgatcctga 841 aaatggtgca gctggactct attgaagatt tggaagtgacttgtacctgg aagctaccca 901 ccttggcgaa attttctcct tacctgggcc agatgattaatctgcgtaga ctcctcctct 961 cccacatcca tgcatcttcc tacatttccc cggagaaggaagagcagtat atcgcccagt 1021 tcacctctca gttcctcagt ctgcagtgcc tgcaggctctctatgtggac tctttatttt 1081 tccttagagg ccgcctggat cagttgctca ggcacgtgatgaaccccttg gaaaccctct 1141 caataactaa ctgccggctt tcggaagggg atgtgatgcatctgtcccag agtcccagcg 1201 tcagtcagct aagtgtcctg agtctaagtg gggtcatgctgaccgatgta agtcccgagc 1261 ccctccaagc tctgctggag agagcctctg ccaccctccaggacctggtc tttgatgagt 1321 gtgggatcac ggatgatcag ctccttgccc tcctgccttccctgagccac tgctcccagc 1381 ttacaacctt aagcttctac gggaattcca tctccatatctgccttgcag agtctcctgc 1441 agcacctcat cgggctgagc aatctgaccc acgtgctgtatcctgtcccc ctggagagtt 1501 atgaggacat ccatggtacc ctccacctgg agaggcttgcctatctgcat gccaggctca 1561 gggagttgct gtgtgagttg gggcggccca gcatggtctggcttagtgcc aacccctgtc 1621 ctcactgtgg ggacagaacc ttctatgacc cggagcccatcctgtgcccc tgtttcatgc 1681 ctaactagct gggtgcacat atcaaatgct tcattctgcatacttggaca ctaaagccag 1741 gatgtgcatg catcttgaag caacaaagca gccacagtttcagacaaatg ttcagtgtga 1801 gtgaggaaaa catgttcagt gaggaaaaaa cattcagacaaatgttcagt gaggaaaaaa 1861 aggggaagtt ggggataggc agatgttgac ttgaggagttaatgtgatct ttggggagat 1921 acatcttata gagttagaaa tagaatctga atttctaaagggagattctg gcttgggaag 1981 tacatgtagg agttaatccc tgtgtagact gttgtaaagaaactgttgaa aataaagaga 2041 agcaatgtga aaaaaaaaaa aaaaaaa Translation ofORF (SEQ ID NO:5): 1 merrrlrgsi qsryismsvw tsprrlvela gqsllkdealaiaalellpr elfpplfmaa 61 fdgrhsqtlk amvqawpftc lplgvlmkgq hlhletfkavldgldvllaq evrprrwklq 121 vldlrknshq dfwtvwsgnr aslysfpepe aaqpmtkkrkvdglsteaeq pfipvevlvd 181 lflkegacde lfsyliekvk rkknvlrlcc kklkifampmqdikmilkmv qldsiedlev 241 tctwklptla kfspylgqmi nlrrlllshi hassyispekeeqyiaqfts qflslqclqa 301 lyvdslfflr grldqllrhv mnpletlsit ncrlsegdvmhlsqspsvsq lsvlslsgvm 361 ltdvspeplq allerasatl qdlvfdecgi tddqllallpslshcsqltt lsfygnsisi 421 salqsllqhl iglsnlthvl ypvplesyed ihgtlhlerlaylharlrel lcelgrpsmv 481 wlsanpcphc gdrtfydpep ilcpcfmpn

1. A method of treatment of a tumour which comprises administering to asubject in need of treatment an effective amount of an inhibitor ofPRAME, in combination with a second agent selected from the group of aninhibitor of HDAC (an HDCAi) and a retinoid, said inhibitor of PRAMEbeing an RNA interference (RNAi)-based inhibitor; said inhibitor of HDACbeing selected from the group consisting of trichostatin A(TSA),-suberoylanilide hydroxamic acid (SAHA), and PXD, 101; whereinsaid tumour overexpresses PReferentially expressed Antigen in MElanoma(PRAME), and wherein said tumour is a melanoma.
 2. The method of claim1, wherein the inhibitor of PRAME is a small interfering RNA (siRNA)that inhibits expression of PRAME.
 3. The method of claim 1, wherein thetumour is insensitive to the HDAC inhibitor administered alone or toretinoic acid administered alone.
 4. The method of claim 1, wherein theadministering occurs after a level of expression of PRAME in the tumorhas been suppressed by medical treatment.
 5. The method of claim 1,wherein administering further comprises pre-determining a level ofexpression of PRAME in the tumor of the subject.
 6. The method of claim1, wherein the tumor that overexpresses PReferentially expressed Antigenin MElanoma (PRAME) is a primary tumor.
 7. The method of claim 1,wherein the tumor that overexpresses PReferentially expressed Antigen inMElanoma (PRAME) is a tumor metastasis.